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Departamento de Producción Agraria, Universidad Pública de Navarra, E-31006 Pamplona, España
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Fluorescence microscopy was used to describe the distribution of nuclei and the organization of the microtubule network in hyphae of Pleurotus ostreatus. Dikaryotic hyphae of P. ostreatus N001 grow by tip extension with two closely spaced nuclei moving slowly forward with the growing hyphal tip. During vegetative growth of the hyphae, cytoplasmic microtubules are found as long filaments oriented longitudinally within fungal hyphae. When the apical cell reaches a length of approximately 150 µm, the two nuclei divide synchronously. Mitosis occurs in association with clamp connection formation, with one of the nuclei dividing in the hook of the developing clamp connection and the other in the main hypha. After mitosis, two daughter nuclei move forward to approximately the center of the apical cell, while the other two move backward to a central position in the subapical cell. Two septa are formed, one in the clamp and the other across the main axis of the hypha to delimit the apical cell. The use of fluorescence microscopy made it possible to examine the changes in the cytoplasmic microtubules, the configuration of the mitotic apparatus, the site of septation and the post-mitotic nuclear migrations during conjugate division in P. ostreatus dikaryotic hyphae.
Key words: immunofluorescence microscopy, microtubules, mitosis, nuclear migration, Pleurotus
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Heath 1995, Steinberg 2000). Basidiomycetes represent a group of fungi different from Ascomycetes in a number of cytological and biochemical parameters, including mitotic nucleus organization (Heath et al 1987) and cell morphogenesis (Moore 1998). In both these processes, cytoskeletal components are involved but the precise mechanisms of cytoskeletal participation are still obscure and further experimental data from different Basidiomycetes species are needed.
Pleurotus ostreatus is an edible Basidiomycete of increasing biotechnological interest due to its ability to degrade both wood and chemicals related to lignin degradation products (Bezalel et al 1997, Giardina et al 1995, Hecker and Roux 1996). Furthermore, this fungus produces secondary metabolites with pharmaceutical applications (Bobek et al 1993, Bobek and Ozdin 1994, Kurashige et al 1997) and some proteins of potential industrial use (Sarkar et al 1997, Shin et al 1997, Wessels 1997). P. ostreatus has been increasingly used in molecular biology and genetics research (Larraya et al 1999a, 2000). Kim et al (2001) recently described one gene encoding a component of the Pleurotus sajor-caju cytoskeleton, namely beta-tubulin. However, there exist few cytological studies on this organism (Kaminskyj et al 1989) and the organization of the cytoskeleton in the hyphae and its functions during the cell cycle have not been described.
The main objective of this work is to show the relationship between cytoplasmic microtubules and the nuclear movements during conjugate division in the dikaryotic hyphae of P. ostreatus. We employed immunofluorescence methods to examine the positioning of the nuclei, the changes in cytoplasmic microtubule organization, the configuration of the mitotic apparatus and the site of septation during the process. In the dikaryotic hyphae of P. ostreatus, as in many other homobasidiomycetes, a lateral outgrowth, called the clamp connection, is formed during synchronous division of the two nuclei in the apical cells. The developing clamp connections are useful indicators of the cell-cycle phase and aid the study of the cytoskeletal elements during nuclear and cell division. The results presented here may complement a general understanding of the cytoskeleton organization during the cell and life cycles in basidiomycetous fungi and may open the door to further genetic and cytological investigations in P. ostreatus, such as the organization of the cytoskeleton during mating, basidium growth and fruit body formation.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, b). Vegetative cultures of dikaryotic mycelium were grown on solid Eger medium (20 g of malt extract, 15 g of agar, 1 liter of water) (Eger 1976) at 24 C in the dark.
Dikaryotic colonies of P. ostreatus N001 originated from a small inoculum were grown on small pieces of dialysis membrane covered by a thin agarose layer, overlying Eger agar plates. For indirect immunofluorescence (IIF) microscopy of microtubules, the cultures were treated as reported by Runeberg et al (1986) with some modifications. After 2 d growth at 24 C in the dark, the membrane-bound colonies were transferred to 3.7% paraformaldehyde in 50 mM sodium phosphate buffer, pH 6.5 containing 5 mM MgCl2 and 5% polyethylene glycol 6000. The hyphae were fixed 1–4 h and then washed for 30 min in 50 mM sodium phosphate buffer containing 5 mM MgCl2. After washing, the colonies were separated in two groups that were submitted to two different cell-wall digestive enzyme treatments. The first group of colonies was transferred to an enzyme solution containing 0.4% (w/v) lysing enzyme (Sigma) in the washing buffer for 10–30 min at 37 C (Tanabe and Kamada 1994). In the second group of colonies, hyphal walls were partially digested by 0.1% (w/v) lysing enzyme in phosphate-buffered saline solution (PBS), pH 5.5 for 10–30 min. After washing, all the colonies were treated with 0.1% Triton X-100 in PBS, pH 7.3 for 1 h and then rinsed with PBS pH 7.3 for 50 min. The hyphae then were exposed to mouse-monoclonal antibodies against chicken 
-tubulin (DM1A) (Neomarkers) at a final concentration of 1:500 for 12–15 h at 4 C. The hyphae were rinsed in 0.1% (w/v) bovine serum albumin in PBS, pH 7.3, with three changes for 30 min and labeled with the secondary antibodies, FITC-conjugated antimouse IgG (Cappel, U.S.A.) at a dilution of 1:100 for 1 h at 24 C. For control experiments, the hyphae were labeled only with the conjugate that gave no detectable staining.
After labeling with the antibodies, the hyphae were washed in PBS, pH 8.9 with five changes for 12 h. The hyphae located in a 1–3 mm wide zone at the edge of the colony were cut out and mounted for microscopical observation. For nucleus and mitochondria staining, the samples were mounted in glycerol:PBS, pH 8.9 (1:1) containing 4', 6-diamidino-2-phenylindole (DAPI, Sigma; 0.1 µg mL-1). For dual labeling of nuclei and septa, the mounting solution contained both DAPI and Fluorescent Brightener 28 (FB28, Sigma). We successfully stained septa with FB28, but somehow it reduced DAPI staining of nuclei and mitochondria. Different concentrations of DAPI and FB28 were compared to optimize nucleus and septum staining in P. ostreatus hyphae. We found that a solution containing DAPI (0.1 µg mL-1) and FB28 (10-4 µg mL-1) resulted in well-labeled cells.
Tubulin fluorescence was recorded from 20 colonies, each containing 100–200 hyphal tips. P. ostreatus hyphae from three different experiments were examined.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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Mitotic stage – Dividing nuclei were recognized easily on the basis of the fluorescence of the spindle microtubules from metaphase to late anaphase. The DAPI staining of nuclei and IIF labeling of the microtubules made it possible to follow the changes in the structure of the chromatin and the spindle through mitosis in both simultaneously dividing nuclei of dikaryotic hyphae. The technique did not let us study the changes that took place in the structure of the nuclear envelope or nucleolus during mitosis, and therefore we cannot comment on these structures. Labeled cells were selected and arranged in a temporal sequence based on the developmental characteristics of nuclei and microtubules. At least five phases were discernible in hyphal cells at the mitotic stage (Figs. 7–18).
Prophase –
The first phase of mitosis was the formation of the clamp connection with the growth of a lateral hook from the central part of the apical cell (Figs. 7, 8, asterisk). The two nuclei of the apical cell usually occurred close to the hook and showed a diffuse staining pattern with DAPI (Fig. 8). IIF labeling for tubulin revealed diffuse tubulin fluorescence around the nuclei and at each nucleus a bright spot that was interpreted to be the spindle pole body (SPB) (Fig. 7, arrows). SPB also has been detected in other fungi as a bright tubulin spot adjacent to the nucleus entering division (Heath 1978).
The changes in the position and the fluorescence pattern of the nuclei were interpreted to be typical of prophase in the fungal mitosis studied. As the division of the nuclei proceeded, the staining of the chromatin increased, due to condensation, and the spindles became visible.
Metaphase – At this phase, the clamp cell grew and the leading nucleus was observed in the developing clamp, whereas the second nucleus remained in the main hypha (Fig. 10). Two small spindles, one at the clamp connection and one at the main hypha, below the clamp, were seen (Fig. 9). The size of both spindles was 1.8 ± 0.2 µm, and they were orientated more or less perpendicular to their respective nuclei. No cytoplasmic fibers could be observed around the nuclei. The condensation of chromatin was completed, judging from the small size and intensive DAPI staining of nuclei (Fig. 10). No individual chromosomes could be distinguished at this or later stages.
Early anaphase – In the next phase regarded as early anaphase, the length of the spindles increased a little, being shorter in the hook (1.9 ± 0.2 µm) than in the hypha (3.1 ± 0.2 µm). The spindles orientated parallel to each other and to the longitudinal axis of the hypha (Fig. 11). Careful comparison of the staining patterns of chromatin with that of the spindle suggested that the spindle lay in the middle of condensed chromatin, except for a very short region at each pole (Figs. 11, 12).
Late anaphase – The chromatin and spindle increased in length (Figs. 13, 14). The length of the spindle varied from 2.5–4 ± 0.3 µm being a little shorter in the hook than in the hypha (Fig. 13). DAPI staining showed a dark line between two condensed chromatin masses (Fig. 14). The dark line was probably a region left after the sister chromatids moved apart to the poles of the spindle.
Polymerization of astral microtubules from the spindle poles was difficult to see. Only by viewing individual focal planes could these short astral microtubules be detected (Fig. 13, arrows). Cytoplasmic microtubules were observed close to the apex, indicating that apical growth probably continues during mitosis (Fig. 13).
Telophase – During early telophase, short astral microtubules were detected at the spindle poles (Fig. 15, arrows). The chromosomes were separated and four groups of chromatin were seen, instead of two single elongating masses (Fig. 16). One set of chromosomes from the division at the clamp migrated from the clamp to the main hypha as the spindles continued to elongate. At late telophase, when the daughter nuclei moved apart farther, the mitotic apparatus disintegrated and only some remnants were observed (not shown).
Post-mitotic nuclear migrations and septum formations – The four incipient nuclei were designated as a-, b-, c- and d-nuclei, depending on their position with respect to each other, as well as their direction of migration (Figs. 15–26). After mitosis, one daughter nucleus from each division (a and b) rapidly moved toward the tip and the other daughter nucleus from the mitosis in the main hypha (c) moved rapidly backward at about the same time (Fig. 18). When the distance between the nuclei increased and the spindles disappeared, numerous cytoplasmic microtubules were detected around the migrating a-, b- and c-nuclei. The orientation of the microtubules along or ending at a place where the plasma membrane is supposed to be suggested that they were associated with this membrane (Fig. 17). The fourth nucleus (d) remained in the clamp for some time after mitosis (Figs. 18–20). Strong tubulin fluorescence was present at the clamp connection, and several fibers surrounded the d-nucleus still enclosed in the hook (Fig. 17). In hyphae where the opening between the hook cell and the subapical cell had formed, thick tubulin tracks passed through the hole (Fig. 19).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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During vegetative growth, dikaryotic hyphae of P. ostreatus N001 are comparable to those of many dikaryotic basidiomycetous fungi, in that the apical cell grows by tip extension until the cell is approximately 150 µm long. In the apical cell of P. ostreatus, the two nuclei move forward, keeping a constant distance from the apex during apical growth. Nuclear movement and positioning are essential processes in fungal development, and a diversity of data have suggested that microtubules and microtubule-dependent motor proteins are involved in nuclear positioning in fungi (Fischer 1999, Fisher et al 2000, Steinberg 2000).
Using IIF microscopy, we observed cytoplasmic microtubules as long filaments oriented longitudinally within the dikaryotic hyphae of P. ostreatus N001. Microtubules were absent in the apex of P. ostreatus hyphae, or at least they were less abundant in the apex than in the shank of the tube. The organization of the interphase microtubule cytoskeleton appeared to be similar to other filamentous fungi, including Basidiomycetes (Grove et al 1970, Howard and Aist 1980, Roberson and Vargas 1994, Runeberg et al 1986, Salo et al 1989, Tanabe and Kamada 1994, Torralba et al 1998). The arrangement of microtubules parallel to the growing axis of the cell probably facilitates polarized cell growth (Howard and Aist 1980, McDaniel and Roberson 2000, Steinberg et al 2001, Straube et al 2003) and aids protein secretion at the apices (Peterbauer et al 1992, Pedregosa et al 1995, Torralba et al 1996, 1998).
Changes in the microtubule network were observed during the cell division in the dikaryotic hyphae of P. ostreatus. In P. ostreatus, the first signs indicating the beginning of the cell division were the growth of the clamp and the disassembly of cytoplasmic microtubules in a region around the nuclei. The depolymerization of the cytoplasmic microtubules during mitosis of P. ostreatus did not extend to the apex, probably allowing the continuation of hyphal extension and secretion at the apices during nuclear division. Microscopical observation of living hyphae of P. ostreatus N001 (not shown) confirmed that extension growth of hyphae continues during conjugate mitosis. The changes in the microtubule network during cell division in P. ostreatus were similar to those described in other Basidiomycetes where conjugate division occurs in association with clamp connection development such as Coprinus cinereus (Tanabe and Kamada 1994), Schizophyllum commune (Raudaskoski et al 1991, Runeberg et al 1986) and Paxillus involutus (Salo et al 1989). In other Basidiomycetes such as Amanita regalis and Suillus bovinus, no clamp connection development occur and nuclear divisions take place in the middle of the apical cell and the associated disassembly of cytoplasmic microtubules often extended to the apex of the hyphae (Salo et al 1989).
The present IIF microscopy study with tubulin antibodies emphasized that the reduction in fluorescence in the cytoplasmic microtubules around the nuclei first was associated with an increase in size of the SPBs and then with the development of the spindles. This suggested that SPBs could nucleate tubulin that originated from depolymerized cytoplasmic microtubules into spindle formation, as it has been proposed in other fungi (Runeberg et al 1986, Salo et al 1989, Torralba et al 1998).
The structure of the spindles in P. ostreatus N001 was similar to that reported in other clamped dikaryotic hyphae of S. commune (Raudaskoski et al 1991, Runeberg et al 1986) and C. cinereus (Tanabe and Kamada 1994) but also had similarities to that described in the filamentous Ascomycete Aspergillus nidulans (Engle et al 1988, Gambino et al 1984, Torralba et al 1998). In P. ostreatus, the spindle in the main hypha was longer than that in the clamp during conjugate division. This means that the two nuclei in the dikaryon P. ostreatus N001 changed position in the apical cell after each conjugate division (Fig. 26). In the beginning of conjugate division, the leading nucleus in the apical cell of the dikaryon always enters the clamp and divides there, while the second nucleus divided in the hypha just beneath the clamp. The longer length of the spindle in the hypha than that in the clamp permited one of the daughters of the nucleus that divides in the hypha (a-nucleus) to travel farther than the daughter of the nucleus that divided in the clamp (b-nucleus). In this way, the two nuclei alternate in position each time a new apical cell is formed. Similar nuclear behavior has been demonstrated in C. cinereus (Iwasa et al 1998).
After telophase, the weak fluorescence of the spindles and the occurrence of numerous cytoplasmic fibers with particular arrangements at each nucleus indicated the disassembly and reassembly of microtubules as at metaphase. The polarity in the arrangement of the cytoplasmic microtubules was probably related to the nuclear movements that bring the nuclei to the center of the apical and subapical cells.
Post-mitotic nuclear migrations have been studied in basidiomycetous fungi for many years (Morris et al 1995, Niederpruem 1971, 1980, Raudaskoski 1998, Rosenberger and Kessel 1968, Ross 1976), but the role of microtubules in this process is still unclear. Some authors have tended to emphasize the potential force-generating role of astral microtubules (Salo et al 1989). However, evidence against a role of the astral microtubules in nuclear movement has been reported in post-mitotic migration of nuclei after conjugate division of C. cinereus (Tanabe and Kamada 1994) and P. ostreatus dikaryons (Kaminskyj et al 1989). Kaminskyj et al (1989) showed independence between the number of astral microtubules and migration rate of nuclei in P. ostreatus. Using IIF microscopy, we observed a small number of short astral microtubules associated with the migrating nuclei in P. ostreatus N001, but these structures could have been lost during chemical fixation. In C. cinereus, mutations in the beta-tubulin gene induced a defect in astral microtubules, but migration of the nuclei in the dikaryon occurred normally. However, septation in the clamp was disturbed in the mutants, suggesting that microtubules might control septation in the clamp (Tanabe and Kamada 1994). In P. ostreatus, we observed many microtubules surrounding the nucleus still enclosed in clamp connection during the time the septa were formed. Further studies are needed to determine if these microtubules are participating in septum growth.
Because astral microtubules appear not to be involved in force production for nuclear migration, what mechanism is responsible? One possible candidate is the population of cytoplasmic microtubules that were laterally associated with the migrating nuclei in P. ostreatus after mitosis. These microtubules oriented along and ending where it was supposed to be located the plasma membrane, suggesting that they were associated with it. Cytoplasmic microtubules have been associated with migrating nuclei in many species of filamentous fungi (Fischer 1999, Hoch et al 1987, McKerracher and Heath 1985, Oakley and Morris 1980, Steinberg and Schliwa 1993, That et al 1988, Xiang and Morris 1999), especially in Basidiomycetes (Kamada et al 1993, 1989, Lehmler et al 1997, Raudaskoski et al 1994, Steinberg et al 2001, Tanabe and Kamada 1994), and there is an emerging consensus that nuclear migration in fungi involve interactions between microtubules and the cell cortex, mediated by a diversity of cortical proteins (Heath et al 2000, Torralba and Heath 2001). Fungal hyphae seem to contain a membrane skeleton similar to that of animal cells, minimally containing F-actin, spectrin and integrin, the organization of which is unknown (Degousée et al 2000, Heath 1995, Heath et al 2000). Proteins specifically able to interact with both microtubules and actin are becoming well characterized in animal cells (Leung et al 1999). In epithelial cells, cytoplasmic dynein has been proposed to mediate interactions between microtubule and actin networks at the cell cortex (Ligon et al 2001). Cytoplasmic dynein is thought to be the major motor for nuclear migration in filamentous fungi (Xiang et al 1994, Morris et al 1995, Steinberg 2000). In budding yeast, it has been suggested that dynamic microtubules interact with cortical dynein, which exerts pulling forces on these microtubules (Carminati and Stearns 1997).
IIF microscopy and related techniques are powerful tools in the study of the structure and functions of the cytoskeleton in filamentous fungi. This work describes for the first time the distribution of nuclei and the organization of the microtubule network in hyphae of P. ostreatus using fluorescence microscopy. Our results illustrate the dynamic complexity of the microtubule network of P. ostreatus during vegetative growth and conjugate mitosis of dikaryotic hyphae and suggest a role of microtubules in nuclear positioning, conjugate division and postmitotic migration of nuclei.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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2 E-mail: gpisabarro{at}unavarra.es
3 Corresponding author. E-mail: lramirez{at}unavarra.es
Accepted for publication June 3, 2003.
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LITERATURE CITED |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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